This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Many papers are published that employ bottom-up proteomics to identify sites of potentail phosphorylation or other posttranslational modification. It is important to know the total ensemble of PTM for a protein at a particular time in the cell cycle. This information cannot be gleaned from the bottom-up strategy. Top down proteomics will be used to interrogate large parts of a protein (>10kDa) to discover the distributions of PTM during on proteins isolated from cells. The workflow will be developed by studying Chk2, an autophosphorylating kinase with more than 30 sites of phosphorylation. We will map out the distribution of phosphorylation from Chk2 captured at various times during the cell cycle.